The present invention relates, in general, to an animal model for chondrodysplasia, and more particularly, to a transgenic mouse model for achondroplasia in which a fibroblast growth factor receptor 3 gene including a G to A point mutation changing Gly to Arg in codon 380 thereof (numbered according to the human sequence) is introduced into the mouse genome.
Achondroplasia is the most common genetic form of osteochondrodysplasia, with an estimated frequency of 1/15000 to 1/77000 births, Achondroplasia is transmitted in an autosomal dominant fashion with complete penetrance, although 80-90% of cases arise from spontaneous mutations (Andersen, 1989, Iannotti, 1994). The clinical features of heterozygous achondroplasia are very consistent among patients, and include proximal shortening of the extremities, midface hypoplasia, narrowing of the spinal column and relative macrocephaly (Rousseau, 1994, Shiang, 1994, Prinos, 1995). Final achondroplasia adult height ranges between 112 to 145 cm.
Histologically, the epiphyseal and growth plate cartilage of achondroplasia patients have a normal appearance (Rimoin, 1970). However, morphometric examinations of such patients revealed that the growth plate is shorter than normal and that the shortening is greater in homozygous than in heterozygous achondroplasia, suggesting a gene dosage effect (Horton, 1988). The intercolumnar matrix of achondroplasia patients is more abundant than normal, and focus of vascularization and transverse tunneling of the cartilage (ingrowth of blood vessels) was observed in some cases. In addition, marked periosteal bone formation was observed (Rimoin, 1970). The underlying mechanism of achondroplasia is believed to be a defect in the maturation of long bones growth plate chondrocytes (Ponseti, 1970, Maynard, 1981, Iannotti, 1994).
Achondroplasia was recently shown to be caused by point mutations in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3, Shiang, 1994). Virtually all patients show either a G to A or a G to C conversion, changing the codon for Gly 380 to Arg. This guanosine 1138 nucleotide has been described as the most mutable nucleotide to date in the human genome (Bellus, 1995). Other mutations that have been described so far lie within the transmembrane domain (Gly 375 to Cys, Superti-Furga, 1995, Ikegawa, 1995) and within the Ig3-TM linker region (Gly 346 to Glu, Prinos, 1995).
FGFR3 is a high-affinity membrane-spanning receptor for fibroblast growth factors (FGFs). The binding of FGF to the extracellular domain of FGFR3, in the presence of heparan sulfate proteoglycans, induces the dimerization of two receptor molecules, allowing transphosphorylation of tyrosines (and possibly threonine and serine residues) within the activation loop of the intracellular tyrosine kinase domains.
Activation loop phosphorylation greatly enhances the ability of FGFR3 to autophosphrylate as well as to phosphorylate substrates which transmit biological signals into the cell leading to cell proliferation, differentiation, angiogenesis, or embryogenesis (Basilico, 1992, Friesel, 1995, Jaye, 1992, Johnson, 1993).
The role of FGFR3 in the growth plate appears to be one of negative regulation of intrinsic growth rates, since mice that are homozygous for FGFR3 null alleles (e.g., by gene knock-out) show kyphosis, scoliosis, overgrowth of long bones and enlargement of the hypertrophic zone of growth plates (Deng, 1996, Colvin, 1996). This phenotype is consistent with a normal role for FGFR3 in restraining chondrocyte proliferation (upper-hypertrophic cells) and final differentiation (lower-hypertrophic cells) at the growth plates of tubular long bones and at the sutures of the skull.
Mutations in FGFR3 and in other fibroblast growth factor receptor genes can also result in other abnormalities, including skeletal and cranial malformation syndromes (Bonaventure, 1996, Muenke, 1995, Park, 1995, Webster, 1997, Lewanda, 1996). Some of the more frequent mutations of FGFRs associated with craniosynostosis and dwarfism syndromes are listed in Table 1, below.
TABLE 1 Some FGFR mutations associated wth craniosynostosis and dwarfism syndromes Mutation Syndrome FGFR 1 Pro252Arg Pfeiffer FGFR2 Tyr105Cys Crouzon Ser252Trp Apert Ser252Phe(CG.fwdarw.TT) Apert Ser252Leu Normal type crouzon 934CGC.fwdarw.TCT(SP.fwdarw.FS) Pfeiffer Pro253Arg Apert Ser267Pro Crouzon Insertion Gly269 Crouzon 982insTGG [insG] Crouzon Cys278Phe Pfeiffer; Crouzon 1037del9 [delHIQ] Crouzon Deletion His287-Gln289 Crouzon Gln289Pro Crouzon Trp290Gly Crouzon Trp290Arg Crouzon Trp290Cys(G.fwdarw.C) Pfeiffer Trp290Cys(G.fwdarw.T) Pfeiffer Lys292Glu Crouzon 1119-3T.fwdarw.G.sup.f Pfeiffer 1119-2A.fwdarw.G.sup.f Pfeiffer; Apert 1119-1G.fwdarw.C.sup.f Pfeiffer Exon III acceptor splice site Pfeiffer Ala314Ser Pfeiffer Asp321Ala Pfeiffer Tyr328Cys Crouzon Asn331Ile Crouzon 1190ins6 [insDA] Crouzon Gly338Arg Crouzon Gly338Glu Crouzon Tyr340His Crouzon Thr341Pro Pfeiffer Cys342Arg Pfeiffer; Crouzon; Jackson-Weiss Cys342Ser(G.fwdarw.C) Pfeiffer; Crouzon Cys342Ser(T.fwdarw.A) Pfeiffer; Crouzon Cys342Tyr Pfeiffer; Crouzon Cys342Trp Crouzon Cys342Phe Crouzon A1a344Ala(G.fwdarw.A).sup.f Crouzon; unclassified Ala344Pro Pfeiffer Ala344Gly Crouzon; Jackson-Weiss Ser347Cys Crouzon Deletion Gly345-Pro361.sup.a Pfeiffer; Crouzon Ser351Cys Unclassified Ser354Cys Crouzon 1245del9[delWLT] Crouzon Val359Phe Pfeiffer 1263ins6.sup.f Pfeiffer Ser372Cys Beare-Stevenson cutis gyrata Tyr375Cys Beare-Stevenson cutis gyrata Gly384Arg Unclassified
 FGFR 3 Arg248Cys Thanatophoric dysplasia type I Ser249Cys Thanatophoric dysplasia type I Pro250Arg Non-syndromic craniosynostosis Gly346Glu Achondroplasia Gly370Cys Thanatophoric dysplasia type I Ser371Cys Thanatophoric dysplasia type I Tyr373Cys Thanatophoric dysplasia type I Gly375Cys Achondroplasia Gly 380 to Arg Achondroplasia Ala391Glu Crouzon with acantbosis; nigricans Asn540Lys Hypochondroplasia Lys650Glu Thanatophoric dysplasia type II Lys650Met Novel skeletal dysplasia Stop807Gly Thanatophoric dysplasia type I Stop807Arg Thanatophoric dysplasia type I Stop807Cys Thanatophoric dysplasia type I
Clinical similarities had already suggested that achondroplasia is part of a continuous spectrum of diseases that are all due to mutations in FGFR3 and share a common defect (McKusick, 1973). the underlying defect in these disorders is a disruption of the normal, regulated proliferation and differentiation of chondrocytes, which takes place at the epiphyseal plates of long bones and base of skull during osteogenesis. They are characterized clinically by skeletal deformities and varying degrees of dwarfism apparent before birth, typically with disproportion between the lengths of the trunk and the limbs (Horton, 1993).
On the mild side of the spectrum is hypochondroplasia, a condition associated with moderate, but variable disproportionate shortness of limbs. The trunk is normal and the face is otherwise unremarkable. The head may be normal or slightly enlarged with mild frontal bossing. The hands and feet tend to be broad and stubby. Radiographically, changes typically seen in achondroplasia are present in very mild degree. Mild shortening of the long bones with slight metaphyseal flaring are observed. The femoral necks are short and broad. The fibulae are disproportionately long, and the ilia are short and square. Shortening of the lumbar pedicles is mild to moderate and the interpediculate distance from L1 to L5 may narrow slightly.
Histologically, the growth plates of hypochondroplasia patients show no consistent microscopic abnormalities (Sillence, 1979). Some patients with hypochondroplasia have been shown to be linked to chromosome 4p, the locus where FGFR3 resides, and to have a mutation within the intracellular kinase domain of the receptor (Asn 540 to Lys, Bellus, 1995). However, other patients were shown not to be linked to chromosome 4p, and thus hypochondroplasia may be determined by mutations in genes other than FGFR3 (Stoilov, 1995).
On the lethal side of the spectrum is thanatophoric dysplasia, a condition that clinically resembles the lethal phenotype of homozygous achondroplasia patients (Stanescu, 1990). Thanatophoric dwarfs exhibit extreme shortening of the limbs, long narrow trunk, markedly narrow thorax, large abdomen, redundancy of skin folds of arms and legs, large head, extreme platyspondyly, and marked midface hypoplasia (Horton, 1993). The severe thoracic and abdominal malformations ultimately cause death through respiratory distress (Shah, 1973, Wynne-Davies, 1985, Webster, 1997). In the few individuals who have survived for several years with medical intervention, there are also deficiencies in central nervous system development (MacDonald, 1989). Consistent with the tissues affected in thanatophoric dysplasia, FGFR3 (c isoform) is primarily expressed in the central nervous system, in prebone cartilage rudiments and at the cartilaginous growth plates of bones (Peters, 1992, Peters, 1993).
Radiologically, features similar to those of homozygous achondroplasia are recognized in thanatophoric dysplasia patients (Langer, 1969). Although the skull is large (large calvariae) with frontal bossing, the facial bones and cranial base are small. The ribs and the scapulae are short. Platyspondyly is severe with wide intervertebral disc spacing, the iliac bones are short and squared, the acetabular roofs are flat, the sacrosciatic notches are short, the femora are shaped like telephone receivers, and the tubular bones are short, bowed, and cupped at their ends (Gorlin, 1997, Horton, 1993). Newborns with thanatophoric dysplasia have been placed in different phenotypic subgroups (Spranger, 1992). The classic form (type I) is characterized by curved short femurs with or without cloverleaf skull, whereas patients of the type II subgroup have slightly longer and straighter femurs and invariably fusion of all cranial sutures, resulting in cloverleaf skull (Young, 1973).
Histologically, in thanatophoric dysplasia there is a generalized disorganization of endochondral ossification at the bone growth plate (Rimoin, 1974), less proliferative and hypertrophic chondrocytes (Horton, 1993, Delezoide, 1997) and discrete areas of short but relatively normal growth plate architecture and fibrotic lesions are observed. These lesions seem to be associated with epiphyseal vascular canals near the growth plate. "Ossification tufts" which are mineralization extensions of the subchondral bone, are also characteristics of the disorder (Horton, 1988).
Thanatophoric dysplasia type I dwarfism has been shown to be caused by mutations at six different sites within FGFR3, but all reported cases of thanatophoric dysplasia type II result from the same Lys 650 to Glu point mutation in the tyrosine kinase domain of FGFR3 (Rousseau, 1995, Tavormina, 1995, Tavormina, 1995, Rousseau, 1996). Phosphorylation of the two activation loop residues corresponding to Tyr 647 and Tyr 648 of FGFR3 has been shown to be essential for activation of the tyrosine kinase activity and the biological activity of the related receptor FGFR1 (Mohammadi, 1996), suggesting that they would also be major sites of activating autophosphorylation in FGFR3. The spectrum of severity observed in patients with FGFR3 mutations is directly related to the degree of receptor activation (Naski, 1996, Webster, 1996).
There is thus a widely recognized need for, and it would be highly advantageous to have, an animal model for chondrodysplasia and in particular a mouse model for achondroplasia in which a mutated FGFR3 gene which causes gain of function is introduced into the mouse genome. Such a model can be exploited to gain better understanding of the disease and as an experimental model with which experimental therapy to chondrodysplasias, such as achondroplasia, can be exercised.